Digital molecular assays

ABSTRACT

Provided herein are systems, devices and methods for the rapid and accurate measurement of analytes by assay of binding events, by direct, digital measurement of individually resolved analyte/reporter binding events. The digital molecular assay systems, devices and methods disclosed herein are capable of particle-by-particle readout using optical reporter molecules that detect and report the binding of a single analyte molecule, and report each such binding in binary format. Such digital molecular assay systems, devices and methods are useful in a variety of applications, such as on mobile electronic devices for use in the field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/493,237, filed Sep. 11, 2019, which is a national stage entry under35 USC § 371 of International Application No. PCT/US2018/022061, filedMar. 12, 2018, which claims the benefit of priority of U.S. ProvisionalApplication No. 62/470,303, filed Mar. 12, 2017, the disclosure of whichis hereby incorporated by reference as if written herein in itsentirety.

Point-of-care diagnostics and other assays performable in the field area pressing need. If the delay and expense associated with sending assayssuch as diagnostic tests, especially blood tests, to dedicatedlaboratories for analysis could be eliminated, responses could be mademore efficiently and effectively. Clinical laboratories deliverdiagnostic tests by performing biochemical assays on precision, benchtopinstruments. Efforts to miniaturize these instruments or replicate theirfunction on mobile electronic devices are fraught with difficulty. Inmany cases the results are unusable.

What are needed are inexpensive, but accurate, point-of-care says suchas diagnostic tests that provide quick and accurate results, for exampledoctors and their patients.

Provided herein are systems, devices and methods for the rapid andaccurate measurement of analytes by assay of binding events, by direct,digital measurement of individually resolved analyte/reporter bindingevents. The digital molecular assay systems, devices and methodsdisclosed herein are capable of particle-by-particle readout usingoptical reporter molecules that detect and report the binding of asingle analyte molecule, and report each such binding in binary format.Such digital molecular assay systems, devices and methods are useful ina variety of applications, such as on mobile electronic devices for usein the field.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a conceptual illustration of an analog assay.

FIG. 2 is a conceptual diagram of analog assay procedures.

FIG. 3 is a simulation of analog assay results.

FIG. 4 is a conceptual diagram of part of a digital molecular assaysystem.

FIG. 5 is a conceptual diagram of part of an alternate digital molecularassay system.

FIG. 6 is a conceptual diagram of digital molecular assay image data.

FIG. 7 is an image of digital molecular assay data.

FIG. 8 is a simulation of digital molecular assay results.

FIG. 9 shows a mobile electronic device with a clip-on assay chipreader.

FIG. 10 illustrates codes that may be embedded in a digital molecularassay.

FIG. 11 shows the effect of variations in reporter volume thickness onreporter molecules. In this FIG. 1 represents the reporter volume, 2represents the surface of a recorder device, 3 represents a reportermolecule, and 4 represents an optical path from reporter molecule torecorder device.

FIG. 12 shows thin reporter volumes and properties thereof. In this FIG.1 represents the reporter volume, 2 represents the surface of a recorderdevice, 3 represents a reporter molecule, 4 represents an optical pathfrom reporter molecule to recorder device, and 5 represents a largerparticle that is attached to, or contains, the reporter molecule 4.

FIG. 13 shows the application of curve-fitting methods to opticalspectra. The horizontal axis refers to wavelength in nm, and thevertical axis refers to the intensity of an optical signal.

FIG. 14 shows binding isotherm plots for a strongly binding reportermolecule (upper curve) and a weakly binding reporter molecule (lowercurve).

FIG. 15 shows the effect of error on the determination of analyteconcentration at low reporter molecule saturation. The horizontal axisis analyte concentration, and the vertical axis is the ratio ofbound/total reporter molecule concentration. The dark horizontal andvertical arrows indicated with 1 represent a “correct” determination ofreporter molecule concentration and analyte concentration. The lighthorizontal and vertical arrows indicated with 2 represent an “incorrect”determination of reporter molecule concentration and analyteconcentration.

FIG. 16 shows the effect of error on the determination of analyteconcentration at high reporter molecule saturation. The horizontal axisis analyte concentration, and the vertical axis is the ratio ofbound/total reporter molecule concentration. The dark horizontal andvertical arrows indicated with 1 represent a “correct” determination ofreporter molecule concentration and analyte concentration. The lighthorizontal and vertical arrows indicated with 2 represent an “incorrect”determination of reporter molecule concentration and analyteconcentration.

DETAILED DESCRIPTION OF THE DISCLOSURE

Although there is great demand for performing biochemical assays onmobile electronic devices, attempts to simply miniaturize conventionalassays and perform them outside the controlled environment ofprofessional, clinical laboratories have not succeeded in the past.Conventional biochemical assays cannot be reliably miniaturized becausethey are inherently analog measurements.

Digital assays eliminate inherent uncertainties of analog assays in atleast three ways: (1) digital assays are based on binary events that arehighly resistant to analog noise; (2) digital assays eliminate errorsoriginating from the unknown fraction of inactive assay molecules in ananalog assay; (3) digital assays eliminate problems associated withspatial inhomogeneity such as non-uniform illumination.

Consider, for example, an antigen-antibody assay designed to measure theconcentration of antigen in a sample that is mixed with a knownconcentration of antibodies. The assay has an optical readout in whichantibodies that bind antigen emit a different optical signal than thosethat do not. (Unbound antibodies might emit no signal, for example)Given the affinity of antigen-antibody binding, a bulk optical readoutsignal may be used to estimate antigen concentration.

This procedure can be made to work reasonably well in a professionallaboratory setting with strict quality controls. It fails miserably,however, when performed with mobile devices in a field setting.Conventional, analog biochemical assays are delicate and give wildlyinaccurate results when performed on cell phones, tablets, and similardevices.

One problem is that without strict laboratory protocols a large fractionof the supposedly known concentration of antibodies may be inactive. Ina field setting anywhere from 10% to 100% of the antibodies may berendered inactive due to improper handling, contamination, denaturationand other problems. Worse, the fraction of inactive antibodies isunknown. It is unobservable and represents a systematic error thatcannot be eliminated by averaging observed data. The fraction of unboundantibodies and the fraction of inactive antibodies is confounded; theirsignals are indistinguishable.

Digital assays reduce or eliminate this problem by counting individualbinding events between analyte and reporter molecule, such as antigenand antibody or complementary nucleotide sequences, rather thanaveraging the results of millions of them. Mobile devices are wellsuited for digital assays because they include high quality camerascapable of sampling millions of biochemical events—as many as one perpixel or tens of millions per exposure. Mobile devices also includesignificant processing power for image analysis and communicationcapabilities for reporting results and offloading processing ifnecessary.

Digital assays select features in an image and classify them as valid ornull. Null features include anything in an image that does not meetspecific criteria for position, brightness, wavelength or shape, forexample. Inactive antibodies are a common source of null features, butirregular sample illumination, imprecise optical alignment, sampleirregularities—all common problems in a mobile setting and in otherscenarios with inadequate controls—also contribute. In a digital assay,null features are discarded for data analysis; only valid featurescontribute to assay results. Valid features are counted as bound orunbound, and those are the only possibilities. Yes or no; one or zero.If, in a digital assay, 463 valid features are counted as bound and 886features are counted as unbound, then the bound fraction is463/(463+886)=463/1349=0.343. This kind of result comes from a digitalprocess. When it is combined with known analyte binding affinity, itprovides the desired analyte concentration. The fraction of eventsclassified as null makes no difference in the result.

It is helpful to keep in mind that it is the assay itself that isdigital. This concept has nothing to do with the ubiquitous digitizingof analog results. Signals produced by analog assays may be digitizedfor analysis or storage, but digitizing an analog signal cannot removesystematic errors that are “baked in” to it. As an analogy, musicalrecordings made with analog equipment retain static pops andhiss—inseparable from the music in an analog recording process—even ifthe recording is stored digitally.

Turning now to the figures, FIG. 1 is a conceptual illustration of ananalog assay. A cuvette contains a sample. The sample may be a solutioncontaining antigen and antibodies, for example. The antibodies may belabeled so that, upon binding an antigen molecule, the newly formedantigen-antibody complex emits an optical signal when interrogated by anoptical excitation. The optical signal may be a spectral measurement;i.e. light intensity versus wavelength. The cuvette, even though it mayhold a small sample volume, just a few milliliters is a common size,contains many billions of antibodies and antigen molecules. The observedspectrum is a composite of spectra emitted by billions of bound, labeledantigen-antibody complexes. But, an unknown fraction of the antibodiesdon't work; they can't bind antigen because they are jammed up inaggregates, denatured or have other problems.

FIG. 2 is a conceptual diagram of analog assay procedures. The assaybegins with an unknown antigen concentration mixed with an antibodyconcentration. The ratio of active antibodies (ready and able to bindantigen) to inactive antibodies (unable or unavailable to bind antigen)is not known. In a professional laboratory setting, trained techniciansfollowing strict procedures in a controlled environment can keep theactive-to-inactive ratio high or at least consistent. In a field orpoint-of-care setting, however the ratio of active-to-inactiveantibodies is much lower and, worse, totally inconsistent.

Active antibodies bind antigen at a rate determined by: theantigen-antibody affinity, the concentration of antigen, and the unknownconcentration of active antibodies.

FIG. 3 is a simulation of analog assay results. The bulk spectrum (heavysolid curve) represents what is observed (“SPECTRUM OUT”) in an analogassay. The numerous, light dashed curves represent spectra from singleantigen-antibody binding events. These spectra are not observable in ananalog assay, however. In the simulation of FIG. 3 , unbound, activeantibodies emit light around 495 nm wavelength while bound, activeantibodies emit light around 505 nm wavelength. An unknown number ofinactive antibodies do not emit light. This means that the bulk spectrumdoes not provide sufficient information to measure the number of boundantibodies as a fraction of all active antibodies.

The situation is worse in an actual experiment because inactiveantibodies may still emit light, but that light provides no informationabout antigen binding. It just contributes to systematic error.

FIG. 4 is a conceptual diagram of part of a digital molecular assaysystem. The digital molecular assay illustrated in FIG. 4 depicts anexample wherein mobile dark-field microscopy performed with a smartphonecamera, for example, captures an image of a plasmonic nanoparticlesandwich-type immunoassay. In this assay, a total internal reflection(TIR) substrate is coated with plasmonic nanoparticles functionalizedwith capture antibodies designed to bind an antigen of interest.Additional plasmonic nanoparticles, functionalized with the same ordifferent antibodies (i.e., designed to bind an equivalent part of theantigen, or a different part) are introduced, along with the antigen ofinterest in a sample. Excitation light is introduced into the substratefrom an edge. Bound antibodies emit different optical signals thanunbound and inactive antibodies. Antibodies may be inactive due to manyfactors, such as degradation or, more commonly, aggregation. Thediffering signals may appear in an image as different sizes, brightness,spectra, shapes, et cetera, and be sorted as active or null consideringone or more of these factors.

FIG. 5 is also a conceptual diagram of part of a digital molecular assaysystem; it is an alternative of the example above wherein the TIRsubstrate is coated with plasmonic nanoparticles functionalized withcapture nucleotide sequences of DNA or RNA, complementary to part of theanalyte DNA/RNA of interest. Additional plasmonic nanoparticles,functionalized with different cDNA/cRNA (i.e., designed to bind anothersection of the analyte DNA/RNA) are introduced, along with the analyteDNA/RNA in a sample. Bound DNA/RNA sequences emit different opticalsignals than unbound and inactive antibodies.

FIG. 6 is a conceptual diagram of digital molecular assay image data;i.e. part of an image captured by a mobile device camera operating as adark field microscope. The image includes spots produced by boundantigen-antibody complexes, spots from unbound antibodies, spots frominactive or null antibodies and a defect zone which may be an area ofthe image that is defective for any of a number of reasons. Illuminationof the image may be non-uniform, even far from uniform. As long as thespatial illumination pattern is known, by recording an image at theillumination wavelength, for example, results at any point in the imagemay be normalized to the known illumination.

FIG. 7 is a depiction of digital molecular assay data obtained by amobile device camera operating as microscope. The figure is lessimpressive when shown in grayscale as it is in this disclosure comparedto the original color image, so it has been manually enhanced forsubmission in black-and-white. White circles have been drawn aroundspots in the image that correspond to active antibodies. All other spotsin the image are null or inactive antibodies. Of the active antibodies,4 out of 11 are bound; these are depicted by way of example with a graycircle around the white one. The identification of active versus null,and bound versus unbound is performed by image analysis software. Imageanalysis may be performed on the mobile device. Alternatively, themobile device may send the image to another processor. It may send theimage to a virtual server in the computer cloud, for example.

As an example of image processing to distinguish bound from unboundantibodies in a digital molecular assay, FIG. 8 is a simulation ofdigital assay results. FIG. 8 represents spectra from six spots in animage from a digital assay. The criteria for bound versus unbound iswhether the spectrum from a spot lies above or below 500 nm inwavelength. Spots with spectra that do not fall in the range shown inthe figure are null. From top to bottom, the spectra correspond to spotsfrom unbound, unbound, bound, inactive (null), unbound and boundantibody sites. There are two positive results, three negative and onenull. Thus the fraction of bound antibodies is ⅖. As mentioned above, anactual experiment is complicated by optical signals from inactiveantibodies. Thus the selection criteria may be more complicated thanspectral center above or below a certain wavelength. The criteria mayinvolve narrow spectral bands, intensity criteria, spectral shape, andspatial shape as examples.

The selection criteria also take into account knowledge of a spatialillumination pattern. Intensity measured at an emission wavelength isnormalized by illumination intensity at an excitation wavelength at thesame location in an image. This eliminates problems of spatialinhomogeneity which plague analog measurements. The assay proceedsdigitally on a particle-by-particle basis considering “EXCITATION IN”and “SPECTRUM OUT” for each particle. The result for a given particlecan only be 0 or 1.

Digital molecular assays may be performed with mobile devices asillustrated in FIG. 9 which shows a mobile electronic device with aclip-on assay chip reader. The assay chip reader may include opticalcomponents that adapt the mobile device camera for dark field imaging,for example. The assay chip is designed to receive analyte solution andmay be pre-coated with antibodies.

Since a digital assay is based on imaging and image analysis, codes maybe placed on an assay chip and read out from the same images used tomeasure binding events. FIG. 10 illustrates codes that may be embeddedin a digital assay. Examples include bar codes, quick response (QR)codes, quantum dots that emit light at engineered wavelengths,nanoparticle reporters of temperature, humidity, light exposure, gasexposure and other environmental data. Identifying marks representingparticular assay types or sample identification may also be included.

The examples discussed above are presented using antigen-antibodybinding. Antigen and antibodies may be linked to optical reportermolecules for assay readout. Assays involving other cross-linkingmechanisms may also be performed digitally. For example, assays based onhybridization of DNA or RNA fragments bound to optical reportermolecules may be performed as digital molecular assays wherecomplementary DNA or RNA fragments take the place of antigen andantibody molecules. As an example, a first part of a short DNA sequencemay be bound to an optical reporter and a second part of the short DNAsequence may be bound to another optical reporter. When the first andsecond part bind to a longer, complementary DNA sequence, the twooptical reporters are brought close together and therefore emit adifferent optical signal compared to when they are farther apart. Thiskind of assay may be used to detect the complementary DNA sequence.

In conclusion, it is a fool's errand to use a mobile device as asurrogate for professional laboratory instrumentation with analogassays. Digital molecular assays allow particle-by-particle readout ofindividual interactions between single analyte molecules and reportermolecules, rendering irrelevant the practical problems attendant withtraditional assays. Leveraging the imaging and image processingcapabilities of mobile devices to provide diagnostic results that reduceor eliminate common sources of systematic errors found in analog assays,digital molecular assays enable in-field assessments such aspoint-of-care diagnostic tests that help doctors and patients obtaincritical health information quickly and inexpensively, and thecollection and analysis of data across a wide range of applications.

Terms and Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the terms below have the meanings indicated.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” or “between n₁ . . . and n₂” is used, where n₁ and n₂ are thenumbers, then unless otherwise specified, this notation is intended toinclude the numbers themselves and the range between them. This rangemay be integral or continuous between and including the end values. Byway of example, the range “from 2 to 6 carbons” is intended to includetwo, three, four, five, and six carbons, since carbons come in integerunits. Compare, by way of example, the range “from 1 to 3 μM(micromolar),” which is intended to include 1 μM, 3 μM, and everythingin between to any number of significant figures (e.g., 1.255 μM, 2.1 μM,2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “accuracy”, as used herein, alone or in combination, is used torefer to the closeness of a reported or estimated value from the truevalue. An inaccurate measurement, observation, or estimation deviatesfrom the true value. An accurate measurement, observation, or estimationdoes not deviate from the true value.

The term “analyte”, or “analyte molecule”, as used herein, alone or incombination, is used to describe a molecule or particle for which thepresence or absence, or amount, in a sample is originally unknown, andfor which knowledge of the presence or absence, or amount, contained ina sample would be useful. Examples of analytes include biomolecules,such as: peptides, proteins, cytokines, and prions; antibodies, andfragments thereof; nucleic acids (DNA/RNA) and particles containingthem, such as histones; small organic and bioinorganic molecules, suchas carbohydrates, lipids, hormones, and intermediates and products ofmetabolism; macromolecules, such as macrocycles, biopolymers (e.g.oligosaccharides, polyphenols, and plastics); and viruses, viralparticles, viral products (e.g. virokines). An analyte may also becategorized as a biomarker, that is, a composition and/or molecule or acomplex of compositions and/or molecules that is associated with abiological state of an organism (e.g., a condition such as a disease ora non-disease state) and can report the presence of disease, injury, orcellular or organismal damage. When such markers bind to an antibody ora fragment thereof, they may be referred to as antigens. Values formeaningful (e.g., normal and abnormal) levels of analytes detected bythe digital molecular assays disclosed herein will be known to those ofskill in the relevant art.

The term “area detector”, as used herein, alone or in combination,refers to a recording device that can record an image from a source,i.e., record not only the intensity of an incoming optical signal, butthe origin of an optical signal. Common examples of area detectors aretelevision cameras, digital SLR cameras, and cellphone cameras.

The term “assay chip”, as used herein, alone or in combination, refersto a microarray of reporter molecules (e.g. optical reporter molecules)spotted or otherwise deposited onto a reporter surface, optionallyenclosed within a relatively thin, flat cuvette such as a slide, whichcan be exposed to a sample containing analyte such that the interactionbetween the capture elements of the optical reporter molecules and theanalyte can be observed. Techniques for the production of assay chipsare known in the art. An assay chip may comprise optical reportermolecules or plasmonic nanoparticles functionalized with antibodies,proteins, DNA, RNA, etc.

The term “assay chip reader”, as used herein, alone or in combination,refers to a system or device for observing and recording signals from anassay chip. An assay chip reader may be part of a digital molecularassay system as disclosed herein, and typically comprises a chamber forreceiving an assay chip, a recording device such as an image sensor(e.g., a camera), a means for transmitting the data collected from theassay to memory, and optionally, a light source such as a light-emittingdiode (LED). Additionally, the assay chip reader may containmicrofluidic hardware such as pumps, channels, chambers for solutions,valves, mixers, and the like; and hardware and/or software forperforming at least some analysis of the data. In certain embodiments,an assay chip reader may be coupled with a smartphone or other mobiledevice and used as part of a portable assay system; miniaturizedmicroplate and chip readers are known in the art.

The term “binding isotherm”, as used herein, alone or in combination,refers to the degree of binding of bound reporter molecules to analytemolecules at different concentrations of analyte. In general, the degreeof binding, which can be defined as the ratio of bound reportermolecules to total reporter molecules, increases with increased analyteconcentration, and eventually approaches 1, as nearly all reportermolecules are bound to analyte molecules.

The term “binning”, as used herein, alone or in combination, refers tothe combination of signals from two or more pixels into one signal.Binning can be used when spatial resolution can be sacrificed in orderto improve signal-to-noise. “2×2 binning”, by way of example, refers tothe grouping of pixels into 2×2 squares, and summing the signals fromthe pixels contained in each square.

The term “biomolecule”, as used herein, alone or in combination,includes any type of organic or bioinorganic molecule for whichdetection (either qualitative or quantitative) may be desired, includingbut not limited to, peptides, proteins, nucleic acids, sugars, mono- andpolysaccharides, lipids, lipoproteins, whole cells, and the like.

The term “camera,” as used herein, refers to a type of image sensor forrecording visual images, for example as digital images. A “megapixelcamera” is a camera that can record one million, or multiples of onemillion, pixels per image. Many smartphone cameras compriseten-megapixel or more cameras.

The term “communication interface,” as used herein, refers to a meansfor transferring data from a device or system as used herein to anotherdevice or system. Examples of wireless communications interfaces includethose used in wireless devices such as mobile phones, for examplecellular, wi-fi, and Bluetooth technologies.

The term “concentration”, as used herein, alone or in combination,refers to the amount of a solute in a solution per unit volume ofsolution. Concentration can be specified in units of molarconcentration, i.e. number of moles of solute per liter of solution, ornumber concentration, i.e., number of molecules of solute per liter ofsolution. Molar concentration and number concentration can be readilyinterconverted. As used herein, the term “concentration” is expanded toinclude systems outside the traditional definition of “solution”, e.g.,systems containing molecules tethered to a solid support.

The term “deconvolution”, as used herein, alone or in combination, isused to describe a method for determining, from a collective opticalsignal that is composed of individual optical signals from a pluralityof optical reporter molecules, the individual optical signals from theindividual optical reporter molecules. Deconvolution can usecurve-fitting techniques to determine the individual spectral featuresfrom individual optical reporter molecules that partially overlap acrossa spectral region and that have combined to form a single collectivespectrum. Deconvolution can use curve-fitting techniques to determineindividual images from individual optical reporter molecules thatpartially overlap in a spatial region of a detector and that havecombined to form a single collective image. It will be understood thatdeconvolution techniques are particularly useful for small groups ofoptical reporter molecules.

The term “detect” or “detection”, as used herein, alone or incombination, is used to describe a method of determination of theexistence, presence, or fact of an analyte in a sample.

The term “divergence” indicates the deviation from perpendicularity thatis accommodated by the recording device. An idealized area-detector typerecording device will accept only light rays that are perpendicular tothe plane of the detector. Actual area detectors will allow light raysthat arrive at an angle from the perpendicular. Although this featurecan increase signal-to-noise (since more light rays are accepted by thedetector), it also decreases spatial resolution, depending on the sizeof the divergence angle allowed, and the size of the area detector pixeland distance between the area detector and the sample plane.

The term “incubate”, as used herein, alone or in combination, is used todescribe a process of exposing reporter molecules to a sample that canpotentially contain an analyte molecule.

The term “oblong” as used herein, alone or in combination, is used todescribe a volume having unequal dimensions. Examples of oblong volumesinclude prisms or cylinders for which the distance between the end facesis either significantly larger or significantly smaller than dimensionsparallel to the end faces. A further example of an oblong volume is anellipsoid for which one axis is either significantly larger orsignificantly smaller than the other axes.

The term “optical path”, as used herein, alone or in combination, isused to describe the path from reporter molecule to detector.

The term “optical reporter molecule,” or, equivalently, “opticalreporter,” as used herein, alone or in combination, is used to describea reporter molecule that is capable of reporting either the presence orabsence, or the amount or concentration of, an analyte molecule, with anoptical signal. The presence or absence of the analyte molecule(optionally, in certain assay formats, with another optical reportermolecule) in contact with the optical reporter molecule, induces achange in the optical signal. An optical reporter molecule bound toanalyte (“bound optical reporter molecule”) will emit a different signalthan an optical reporter molecule not bound to analyte (“unbound opticalreporter molecule”).

The term “optical signal”, as used herein, alone or in combination, isused to describe a signal that originates from an optical reportermolecule. The optical signal may fall in the visible range of thespectrum, or outside the visible range of the spectrum. The signal maybe, for example:

-   -   wavelength of light;    -   intensity of signal;    -   brightness;    -   the shape of a signal or spectrum;    -   the presence or absence of spectral bands;    -   the extinction coefficient of an absorption band;    -   the λ_(max) of an absorption band;    -   the quantum yield of an emission band; or    -   the fluorescence anisotropy of an emission band.        The optical signal from an optical reporter molecule may change        upon binding of an analyte molecule. The change in optical        signal upon binding may be one of the following:    -   a shift in the center of a spectrum above or below a specified        wavelength;    -   a shift in wavelength of maximum intensity (λ_(max));    -   a change in the size or intensity of the signal;    -   an increase or decrease in brightness;    -   a change in the shape of the signal;    -   the presence or absence of spectral bands;    -   a change in shape of a spectrum;    -   a change in the extinction coefficient of an absorption band;    -   a change in the λ_(max) of an absorption band;    -   a change in the quantum yield of an emission band; and    -   a change in the fluorescence anisotropy of an emission band.

The term “pixel”, as used herein, alone or in combination, refers to anarea on an area detector, for example an image sensor, whose signal canbe measured independently from other pixels. Area detectors are commonlydivided into a two-dimensional grid of pixels, with the size of eachpixel, and the count of pixels in the two directions, determined by thearea detector manufacturer.

The term “precision”, as used herein, alone or in combination, is usedto refer to the estimate of error that is associated with a reported orestimated value. A low precision measurement, observation, or estimationis associated with a high degree of uncertainty about the closeness ofthis number to the actual value. A high precision measurement,observation, or estimation is associated with a low degree ofuncertainty about the closeness of this number to the actual value.Precision can often be quantified by the use of error bars on graphs orranges for numerical values. For example, an estimated value that isreported as 10.5±0.1 indicates that the true value is very likelybetween 10.4 and 10.6; with a small but nonzero chance that the truevalue is outside this range.

The terms “protein,” “polypeptide,” “peptide,” and “oligopeptide,” areused interchangeably herein and include any composition that includestwo or more amino acids joined together by a peptide bond. It will beappreciated that polypeptides can contain amino acids other than the 20amino acids commonly referred to as the 20 naturally occurring aminoacids. Also, polypeptides can include one or more amino acids, includingthe terminal amino acids, which are modified by any means known in theart (whether naturally or non-naturally). Examples of polypeptidemodifications include e.g., by glycosylation, orother-post-translational modification. Modifications which can bepresent in polypeptides of the present disclosure, include, but are notlimited to: acetylation, acylation, ADP-ribosylation, amidation,covalent attachment of flavin, covalent attachment of a heme moiety,covalent attachment of a polynucleotide or polynucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphatidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

The term “qualitative analysis”, as used herein, alone or incombination, is used to describe a method for determining the absence orpresence of an analyte molecule in a sample. In some embodiments, aqualitative analysis method reports the presence or absence of a singlemolecule of analyte in a sample. In some embodiments, a qualitativeanalysis method incorrectly reports the absence of analyte in a samplethat contains analyte at a level below a certain threshold.

The term “quantitative analysis”, as used herein, alone or incombination, is used to describe a method for determining the amount ofan analyte molecule in a sample.

The term “recording device”, as used herein, alone or in combination,refers to a device for recording an optical signal. In certainembodiments, the optical signal is converted to an electrical signal. Incertain embodiments, the recording device is a charge-coupled device(“CCD”). In certain embodiments, the recording device is a complementarymetal-oxide semiconductor (“CMOS”) device.

The term “reporter molecule”, as used herein, alone or in combination,is used to describe a molecule that can report either the presence orabsence, or the amount or concentration of, an analyte molecule, andalone or in combination with another reporter molecule, produce adetectable signal in a digital molecular assay. Typically, a reportermolecule will bind to an analyte molecule, and the reporter molecule andanalyte molecule complex will differ significantly in one or morespectral properties. Reporter molecules can be antibodies or fragmentthereof, nucleic acids, proteins, and peptides, any of which may bechemically or biochemically modified. Reporter molecules can also bechimeric molecules comprising a moiety of biochemical origin and asynthetic moiety; examples include an antibody-functionalized plasmonicnanoparticle and a nucleotide-functionalized plasmonic nanoparticle.Reporter molecules can be aptamers based on either nucleic acids orpeptides.

The term “reporter volume”, as used herein, alone or in combination, isused to describe the volume of the measurement device in which thereporter molecules are located. The reporter volume may be substantiallythe same as the sample compartment, or the reporter volume may besmaller. In certain embodiments, the dimension of the reporter volumethat is parallel to the optical paths for the reporter molecules will besmall. In certain embodiments, the reporter volume will constitute amonolayer.

The term “sample”, as used herein, alone or in combination, is used todescribe a composition that contains the analyte of interest. A samplewill often be in fluid, e.g. aqueous, solution. A sample may be chemicalor biological. Blood, plasma, water from a source to be tested, extractsfrom plant, animal, or human tissue samples, are examples of biologicalsamples. A chemical sample would be one that did not contain material ofbiological origin, such as a water sample containing petrochemical orindustrial waste. Biological samples drawn from an organism can include,but are not limited to, the following: blood, serum, plasma, urine,mucus, saliva, sputum, stool, and other physiological secretions, aswell as extracts of tissues, and/or any other constituents of the bodywhich can contain the target particle of interest. Other similarspecimens such as cell or tissue culture or culture broth are also ofinterest.

A biological sample may be fresh or stored (e.g. blood or blood fractionstored in a blood bank). The biological sample may be a bodily fluidexpressly obtained for the assays of this invention or a bodily fluidobtained for another purpose which can be sub-sampled for the assays ofthis invention. In one embodiment, the biological sample is whole blood.Whole blood may be obtained from the subject using standard clinicalprocedures. In another embodiment, the biological sample is plasma.Plasma may be obtained from whole blood samples by centrifugation ofanti-coagulated blood. Such process provides a buffy coat of white cellcomponents and a supernatant of the plasma. In another embodiment, thebiological sample is serum. Serum may be obtained by centrifugation ofwhole blood samples that have been collected in tubes that are free ofanti-coagulant. The blood is permitted to clot prior to centrifugation.The yellowish-reddish fluid that is obtained by centrifugation is theserum. In another embodiment, the sample is urine. The sample may bepretreated as necessary by dilution in an appropriate buffer solution,heparinized, concentrated if desired, or fractionated by any number ofmethods including but not limited to ultracentrifugation, fractionationby fast performance liquid chromatography (FPLC), or precipitation ofapolipoprotein B containing proteins with dextran sulfate or othermethods. Any of a number of standard aqueous buffer solutions atphysiological pH, such as phosphate, Tris, or the like, can be used.

The term “saturation”, as used herein, in reference to bindingphenomena, refers to a state in which nearly all reporter molecules arebound to analyte molecules. A characteristic of a condition ofsaturation is that an increase in the concentration of analyte causes asmall increase in the degree of binding of reporter molecules.

The term “smartphone” as used herein, refers to a handheld personalcomputer with a mobile operating system and an integrated mobilebroadband cellular network connection for voice, SMS, and internet datacommunication, and, typically, wi-fi.

The terms “tablet computer” or “tablet,” as used herein, refers to athin, flat, portable personal computer, typically with a mobileoperating system, LCD touchscreen display, a rechargeable battery, and awireless (optionally, cellular) communication interface.

Embodiments

The invention is further described by the following embodiments.

Embodiment 1. The disclosure provides a method for determining thepresence or concentration of at least one analyte in a sample,comprising:

-   -   in an image of a plurality of signals emitted by at least one        type of optical reporter molecules incubated with at least one        type of analyte molecules;    -   for each type of optical reporter molecules, determining the        number of discrete optical reporter molecules bound to analyte        molecules (“bound optical reporter molecules”) and the number of        discrete optical reporter molecules unbound to analyte (“unbound        optical reporter molecules”) in the image by individually        resolving bound and unbound optical reporter molecules; and,    -   determining the presence or concentration of analyte from the        number of bound optical reporter molecules as a fraction of, or        as proportional to a fraction of, the total number of optical        reporter molecules.

Embodiment 2. In certain embodiments, the disclosure provides a methodfor determining the presence or concentration of at least one analyte ina sample, comprising:

-   -   in an image of a plurality of signals emitted by at least one        type of optical reporter molecules incubated with at least one        type of analyte molecules,    -   for each type of optical reporter molecules, determining the        number of discrete optical reporter molecules bound to analyte        molecules (“bound optical reporter molecules”) and the number of        discrete optical reporter molecules unbound to analyte (“unbound        optical reporter molecules”) in the image by:        -   in certain regions of the image, individually resolving            bound and unbound optical reporter molecules, and        -   in certain other regions of the image, wherein a group of            two or more optical reporter molecules are not resolved,            performing a computational or mathematical deconvolution            that provides the number of bound optical reporter molecules            and unbound reporter molecules in the group; and    -   determining the presence or concentration of analyte from the        number of bound optical reporter molecules as a fraction of, or        as proportional to a fraction of, the total number of optical        reporter molecules.

Embodiment 3. The method of either of embodiments 1 or 2, wherein theoptical reporter molecules are arrayed on a reporter surface.

Embodiment 4. The method of embodiment 3, wherein the optical reportermolecules are arrayed randomly.

Embodiment 5. The method of embodiment 3, wherein the optical reportermolecules are arrayed in a pattern.

Embodiment 6. The method of any of embodiments 1-5, wherein the fractionof bound optical reporter molecules is determined from the number ofunbound optical reporter molecules recorded prior to introduction of thesample.

Embodiment 7. The method of any of embodiments 1-6, wherein theconcentration of the at least one analyte is determined.

Embodiment 8. The method of any of embodiments 1-7, wherein the sampleis a biological or chemical sample.

Embodiment 9. The method of any of embodiments 1-8, wherein the analyteis chosen from:

-   -   a nucleotide sequence; and    -   an antigen.

Embodiment 10. The method of any of embodiments 1-9, wherein the opticalreporter molecule comprises a capture element chosen from:

-   -   one or more nucleotide sequences binds the analyte; and    -   an antibody or a fragment thereof that binds the analyte.

Embodiment 11. The method of any of embodiments 1-10, wherein eachoptical reporter molecule comprises a plasmonic nanoparticle.

Embodiment 12. The method of any of embodiments 1-11, wherein theoptical reporter molecule comprises one or more nucleotide sequencesfunctionalized onto one or more plasmonic nanoparticles.

Embodiment 13. The method of any of embodiments 1-11, wherein theoptical reporter molecule comprises one or more antibodiesfunctionalized onto one or more plasmonic nanoparticles.

Embodiment 14. The method of any of embodiments 1-13, wherein the signalfrom the optical reporter molecule is chosen from:

-   -   wavelength of light;    -   intensity of signal;    -   brightness;    -   the shape of a signal or spectrum; and    -   the presence or absence of spectral bands.

Embodiment 15. The method of any of embodiments 1-14, wherein one signalis produced upon binding of analyte to the optical reporter molecule.

Embodiment 16. The method of any of embodiments 1-15, wherein anothersignal is produced upon binding of analyte to the optical reportermolecule and binding of a second reporter molecule to the analyte.

Embodiment 17. The method of any of embodiments 1-16, wherein thesignals produced by the bound optical reporter molecule and the unboundoptical reporter molecule are different.

Embodiment 18. The method of any of embodiments 1-17, wherein the boundand unbound optical reporter molecules are individually resolved by:

-   -   a shift in the center of a spectrum above or below a specified        wavelength;    -   a change in the size or intensity of the signal;    -   an increase or decrease in brightness;    -   a change in the shape of the signal;    -   the presence or absence of spectral bands; and    -   a change in shape of a spectrum.

Embodiment 19. The method of any of embodiments 1-18, wherein the signalemitted by the optical reporter molecule is wavelength of light.

Embodiment 20. The method of any of embodiments 1-19, wherein the boundand unbound optical reporter molecules are individually resolved by ashift in the center of a spectrum above or below a specified wavelength.

Embodiment 21. The method of any of embodiments 1-20, wherein at leastsome of the optical reporter molecules are affixed to a surface (thereporter surface) such that each affixed optical reporter molecule isspatially resolvable.

Embodiment 22. The method of embodiment 21, wherein the affixed opticalreporter molecules are arrayed in a grid or an approximation thereof.

Embodiment 23. The method of embodiment 21, wherein each affixed opticalreporter molecules is resolvable as one pixel of a recording device.

Embodiment 24. The method of any of embodiments 1-23, wherein activeoptical reporter molecules and inactive optical reporter molecules emitdifferent optical signals.

Embodiment 25. The method of any of embodiments 1-24, wherein the methoddetermines the number of discrete active optical reporter moleculesbound to analyte molecules (“bound active optical reporter molecules”)and the number of discrete optical reporter molecules unbound to analyte(“unbound active optical reporter molecules”) in the image.

Embodiment 26. The method of any of embodiments 1-25, whereinnon-uniform illumination of the sample does not affect the determinationof the presence or concentration of analyte.

Embodiment 27. The method of any of embodiments 1-26, wherein the imageis recorded at a known illumination wavelength.

Embodiment 28. The method of any of embodiments 1-27, wherein results atany point in the image are normalized to the known illumination.

Embodiment 29. The method of any of embodiments 1-28, wherein intensitymeasured at an emission wavelength is normalized by illuminationintensity at an excitation wavelength at the same location in an image.

Embodiment 30. The method of any of embodiments 1-29, wherein defects inone or more sections of the sensor which recorded the image do notaffect the determination of the presence or concentration of analyte.

Embodiment 31. The method of any of embodiments 1-30, wherein one typeof optical reporter molecule is used.

Embodiment 32. The method of any of embodiments 1-31, wherein more thanone type of optical reporter molecule is used.

Embodiment 33. The method as recited in any of embodiments 1-32, whereinthe method employs a sandwich-type assay.

Embodiment 34. The method of any of embodiments 1-33, wherein a firsttype of optical reporter molecules are affixed to a surface (thereporter surface) such that each affixed optical reporter molecule isspatially resolvable.

Embodiment 35. The method of any of embodiments 1-34, wherein the firsttype of optical reporter molecules comprises a capture element for ananalyte functionalized onto a plasmonic nanoparticle.

Embodiment 36. The method of any of embodiments 1-35, wherein a secondtype of optical reporter molecules are added with or after the sample.

Embodiment 37. The method of any of embodiments 1-36, wherein the secondtype of optical reporter molecules comprises a capture element for theanalyte functionalized onto a plasmonic nanoparticle.

Embodiment 38. The method of any of embodiments 1-37, wherein theanalyte is an antigen.

Embodiment 39. The method of embodiment 38, wherein each opticalreporter molecule comprises as the capture element an antibody or afragment thereof.

Embodiment 40. The method of any of embodiments 1-37, wherein theanalyte is a nucleotide sequence.

Embodiment 41. The method of embodiment 40, wherein each opticalreporter molecule comprises as the capture element one or morenucleotide sequences complementary to the analyte nucleotide sequence.

Embodiment 42. The method of any of embodiments 1-41, wherein the methodis performed on a digital molecular assay system comprising a mobiledevice.

Embodiment 43. The method of any of embodiments 1-41, performed on thedigital molecular assay system of any of embodiments 50-70.

Embodiment 44. A method for determining the presence or concentration ofantigen in a sample, comprising:

-   -   in an image of a plurality of signals emitted by at least one        type of optical reporter molecules comprising antibodies        incubated with antigen, determining the number of discrete        active antibodies bound to antigen (“bound active antibodies”)        and the number of discrete active antibodies unbound to antigen        (“unbound active antibodies”) in the image by individually        resolving bound and unbound optical reporter molecules; and,    -   determining the presence or concentration of antigen from the        number of bound active antibodies as a fraction of, or as        proportional to a fraction of, the total number of active        antibodies.

Embodiment 45. The method of embodiment 44, comprising the limitationsof any of embodiments 3-11 and 13-39.

Embodiment 46. The method of embodiment 45, performed on the digitalmolecular assay system of any of embodiments 50-70.

Embodiment 47. A method for determining the presence or concentration ofa target nucleotide sequence in a sample, comprising:

-   -   in an image of a plurality of signals emitted by:    -   a) an optical reporter molecule comprising a first capture        nucleotide sequence complementary to a first part of the target        nucleotide sequence, and    -   b) the first optical reporter molecule comprising the first        capture nucleotide sequence complementary to part of the target        nucleotide sequence and a second optical reporter molecule        comprising a second capture nucleotide sequence complementary to        a second part of the target nucleotide sequence bound to the        target nucleotide sequence (“bound complexes”),    -   determining the number of discrete target nucleotide sequences        bound to optical reporter molecules comprising the first and        second parts of the complementary nucleotide sequence (“bound        complexes”);    -   determining the presence or concentration of the target        nucleotide sequence as a fraction of, or as proportional to        number of bound complexes as a fraction of the total number of        optical reporter molecules emitting detectable signals.

Embodiment 48. The method of embodiment 47, comprising the limitationsof any of embodiments 3-12 and 13-37.

Embodiment 49. The method of embodiment 48, wherein the system is of anyof embodiments 50-70.

Embodiment 50. A digital assay system for determining a concentration ofanalyte in a sample, comprising:

-   -   an image sensor;    -   a screen capable of displaying an image;    -   a microprocessor;    -   memory;    -   image analysis software stored in the memory and executable by        the processor capable of analyzing the data captured by the        image sensor and digitally classifying data; and    -   optionally, a communication interface.

Embodiment 51. The digital assay system of embodiment 50, wherein theimage sensor is capable of operating as part of a dark-field microscope.

Embodiment 52. The digital assay system of embodiment 51, wherein theimage sensor is a megapixel camera.

Embodiment 53. The digital assay system of any of embodiments 49-52,wherein the image sensor is complementary metal-oxide semiconductor(CMOS) camera.

Embodiment 54. The digital assay system of any of embodiments 49-53,additionally comprising a source of light or other electromagneticradiation.

Embodiment 55. The digital assay system of any of embodiments 49-54,wherein the light source comprises a light-emitting diode (LED).

Embodiment 56. The digital assay system of any of embodiments 49-55,additionally comprising a sample chamber that is optionally removable.

Embodiment 57. The digital assay system of any of embodiments 49-56,additionally comprising:

-   -   a reporter surface made of glass or polymer, to one side of        which optical reporter molecules comprising plasmonic        nanoparticles functionalized with capture elements have been        affixed; and    -   a waveguide that is suitable for dark-field microscopy in        contact with the opposite side of the reporter surface.

Embodiment 58. The digital assay system of any of embodiments 49-57,wherein each affixed optical reporter molecule is spatially resolvable.

Embodiment 59. The digital assay system of embodiment 58, wherein theaffixed optical reporter molecules are arrayed in a grid or anapproximation thereof.

Embodiment 60. The digital assay system of embodiment 58 or 59, whereineach affixed optical reporter molecules is resolvable as one pixel of arecording device.

Embodiment 61. The digital assay system of any of embodiments 49-60,wherein the capture element is chosen from:

-   -   one or more nucleotide sequences binds the analyte; and    -   an antibody or a fragment thereof that binds the analyte.

Embodiment 62. The digital assay system of any of embodiments 49-61,wherein the analyte is an antigen.

Embodiment 63. The digital assay system of any of embodiments 49-62,wherein each optical reporter molecule comprises as the capture elementan antibody or a fragment thereof

Embodiment 64. The digital assay system of any of embodiments 49-63,wherein the analyte is a nucleotide sequence.

Embodiment 65. The digital assay system of any of embodiments 49-64,wherein each optical reporter molecule comprises as the capture elementone or more nucleotide sequences complementary to the analyte nucleotidesequence.

Embodiment 66. The digital assay system of any of embodiments 49-65,wherein the microprocessor, memory, image sensor, software, screencapable of displaying an image, and communication interface are allcomprised within a single, portable device.

Embodiment 67. The digital assay system of any of embodiments 49-66,wherein the communication capability is wireless.

Embodiment 68. The digital assay system of any of embodiments 49-67,wherein the single, portable device is chosen from a smartphone and atablet computer.

Embodiment 69. The digital assay system of any of embodiments 49-68,wherein the single, portable device is a smartphone.

Embodiment 70. The digital assay system of any of embodiments 49-69,additionally comprising a case for positioning the smartphone, samplechamber, and light source in close and stable proximity.

Also provided are devices comprising the elements above.

Embodiment 71. Also provided is a digital assay system of any ofembodiments 50-70, which can perform the method of any of embodiments1-41, 44, 45, 47, and 48.

Embodiment 72. A method for performing a biochemical assay comprising:

-   -   incubating antibodies with antigen;    -   obtaining an image of a plurality of the antibodies;    -   classifying the antibodies seen in the image as either active or        null;    -   classifying active antibodies as either bound or unbound;    -   determining the number of bound and unbound antibodies in the        image; and,    -   measuring a concentration of antigen from the number of bound        antibodies as a fraction of the number of active antibodies.

Embodiment 73. The method of embodiment 72, where the antibodies areattached to a surface.

Embodiment 74. The method of embodiment 72, wherein the antibodies andantigen are labeled with optical reporter molecules.

Embodiment 75. The method of embodiment 72, wherein the image isobtained with a mobile device camera.

Embodiment 76. A method for performing a biochemical assay comprising:

-   -   incubating optical reporter molecules bound to a first part of a        short DNA sequence with optical reporter molecules bound to a        second part of the short DNA sequence;    -   obtaining an image of a plurality of the optical reporter        molecules;    -   classifying molecular complexes seen in the image as either        active or null;    -   classifying active molecular complexes as either bound or        unbound;    -   determining the number of bound and unbound complexes in the        image; and,    -   measuring a concentration of a DNA sequence complementary to        both the first and second parts of the short DNA sequence from        the number of bound complexes as a fraction of the number of        active complexes.

Embodiment 77. The method of embodiment 76, where the short DNAsequences are bound to a surface.

Additional embodiments of the above are detailed below.

Applications

The digital molecular assay methods, systems, and devices disclosedherein are useful in a variety of fields and applications. Inparticular, digital molecular assays would be useful in “the field,”that is, in a portable setting. For example, digital molecular assayswould be useful in medical assessment and diagnostics and detection ofpathogens, particularly in remote areas, areas that are underserved ordifficult to access (e.g. due to violent conflict), areas affected by anepidemic, and in other instances where access to traditional assayequipment and/or professionals is limited. They would also be usefulwithin a hospital or clinic, or in a home-visit setting, where theycould be performed or used at point of care or bedside.

Digital molecular assays would be equally useful in a veterinary settingas in a medical, whether in a veterinary office, on a ranch or farm, oranywhere animals in need of testing are located. They could also be usedin horticultural or agricultural applications to test plants or soil forpathogens or symbiotic microorganisms, or detect other genotypes andphenotypes of interest.

Digital molecular assays could also be used to test water forcontamination, e.g., by bacteria, algae, or fungi, or the toxic productsthereof; by petroleum or its products and by-products, and industrialwaste. Such assays would be useful for food safety testing and foragricultural uses, such as field or processing facility testing forpathogens, toxins, adulterants, contaminants, and pests.

Assays

Many types of biochemical assays are adaptable to the digital molecularassay format disclosed herein. Examples include: immunoassays in whichcapture and binding of an antigen by an antibody or a fragment thereof;hybridization assays in which one or more segments of DNA or RNAcomplementary to analyte DNA/RNA of interest is used to capture theanalyte; and ligand binding assays in which a binding partner to areceptor, enzyme, or other protein, or vice versa, is used as thecapture agent for the partner analyte (e.g., protein or fragmentthereof).

It will be appreciated that immunoassays and hybridization assays canemploy a sandwich format in which binding partner pairs, e.g. antibodiesor cDNA/RNA, to the same analyte molecule, e.g., an antigen or targetDNA/RNA, are used. The disclosure thus encompasses binding partnerpairs, e.g., antibodies, wherein both antibodies are specific to thesame molecule, e.g., the same antigen, and wherein one or both membersof the pair comprises an optical reporter molecule as described herein.The combination of multiple capture and reporter elements stillcomprises a signal-producing arrangement which, while comprisingmultiple optical reporter molecules, may still itself be termed anoptical reporter molecule.

Capture binding partners and detection binding partner pairs, e.g.,capture and detection antibody or nucleotide pairs, can be used in thereporter molecule. Thus, although the digital molecular assays disclosedherein allow for label-free detection of analytes, in some embodiments,a heterogeneous assay protocol is used in which, typically, two bindingpartners, e.g., two antibodies or two sequences of DNA or RNA, are used.One binding partner is a capture partner, usually immobilized on a solidsupport such as a plasmonic nanoparticle, and the other binding partneris a detection binding partner, typically with a detectable labelattached, such as another plasmonic nanoparticle. Antibodies andantibody pairs are commercially available, and can also be designed andprepared by methods well-known in the art.

Reporter molecules can be attached to a reporter surface, either bynonspecific adsorption, or by specific covalent linkage. The loading ofreporter molecules will be determined to a large part by theconcentration of the reporter molecules in the preparation solution.More concentrated solutions will provide a higher density of reportermolecules, while at the same time increasing the count of clusters ofreporter molecules that contain two or more particles. This lattereffect is by no means fatal to successful operation: smaller clusters ofreporter molecules can be analyzed with curve-fitting techniquesdiscussed below, while larger clusters that are not suitable for thesetechniques can be flagged as inactive. Considering the conflicting goalsof increasing reporter molecule count and maintaining a manageably smallnumber of reporter molecule clusters, a loading of a maximum of about 1reporter molecule per square micron will prove to be optimal in certainembodiments. For single-molecule detection, a density that would equateto no more than one analyte molecule (bound to optical reportermolecule) per pixel would be useful.

Analysis using sandwich assays can be performed with a multi-stepprocedure: the reporter surface that has been functionalized withcapture molecules is exposed to the analyte. A certain fraction ofcapture molecules will bind to analyte, depending on analyteconcentration. In a second step, the reporter surface is exposed to asolution with detection molecules. Only those capture molecules thathave bound to an analyte in the first step will bind to detectionmolecules in the second step. A clear advantage of this method is thatthe capture and detection molecules can be chosen so as to optimize theoptical signal that is delivered from the capturemolecule/analyte/detection molecule assembly, as compared to the unboundcapture molecule.

The methods disclosed herein can be used to identify a phenotypic orgenotypic state of interest associated with a clinically diagnoseddisease state. Such disease states include, for example, cancer,cardiovascular disease, inflammatory disease, autoimmune disease,neurological disease, infectious disease and pregnancy relateddisorders. Alternatively, states of health can be detected usingmarkers.

The methods disclosed herein can be used to detect genetic variation.The genetic variation herein may include, but is not limited to, one ormore substitution, inversion, insertion, deletion, or mutation innucleotide sequences (e.g., DNA and RNA) and proteins (e.g., peptide andprotein), one or more microdeletion, one or more rare allele,polymorphism, single nucleotide polymorphism (SNP), large-scale geneticpolymorphism, such as inversions and translocations, differences in theabundance and/or copy number (e.g., copy number variants, CNVs) of oneor more nucleotide molecules (e.g., DNA), trisomy, monosomy, and genomicrearrangements. In some embodiments, the genetic variation may berelated to metastasis, presence, absence, and/or risk of a disease, suchas cancer, pharmacokinetic variability, drug toxicity, adverse events,recurrence, and/or presence, absence, or risk of organ transplantrejection in the subject. For example, copy number changes in the HER2gene affect whether a breast cancer patient will respond to Herceptintreatment or not. Similarly, detecting an increase in copy number ofchromosome 21 (or 18, or 13, or sex chromosomes) in blood from apregnant woman may be used as a non-invasive diagnostic for Down'sSyndrome (or Patau's Syndrome or Edwards' Syndrome) in an unborn child.An additional example is the detection of alleles from a transplantedorgan that are not present in the recipient genome-monitoring thefrequency, or copy number, of these alleles may identify signs ofpotential organ rejection.

Measurement Devices and Systems

The digital molecular assay methods described herein employ ameasurement device or system, either of which comprises the partsrequired for analysis of samples. The measurement device contains asample compartment, into which samples are introduced, either by directaddition of the sample of interest, or by insertion of a cuvette orslide, which itself contains the sample of interest. The samplecompartment further provides a component that contains reportermolecules, whose function is to bind to the analyte of interest andproduce an optical signal. For designs which rely on emission methods,the measurement device provides an illumination device for excitation ofchromophores contained in the reporter molecules. The measurement devicecontains a recording device (e.g. an image sensor, e.g. a digitalcamera), which detects and records the optical signal from the reportermolecules. Finally, the measurement device can contain additionalcomponents, such as controls for operation, a device for displaying orreporting analysis results, and an interface with an external computer.The presence of the various optional components, and their specifics,may differ among various designs of measurement devices.

The system or device as a whole can incorporate a mount for orientingthe device for convenient sample addition or removal. The system can becoupled to a mobile computing device. The mobile computing device couldbe a smartphone, handheld computer, tablet computer, or a similarportable computing device. In some examples, the mobile computing deviceincludes all necessary components, such as: a display, a processor, amemory, and program instructions stored in the memory and executable bythe processor, to enable highly automated performance of steps such as:(i) introduction of sample, (ii) optical excitation, (iii) optionalpre-screening of the sample to evaluate sample quality and optimalexposure time, (iv) recording of an image by the recording device, (v)subtraction of detector bias, if required, (vi) digitization of detectorsignal, (vii) recording of digital signal in nonvolatile memory, (viii)recycling of detector, if needed, and (ix) processing of digital signal.The functions could further include determining the result of thedigital assay, and conveying the result in visual form to the end user.

The use of a smartphone or other mobile computing device as thedetection instrument for digital molecular assays allows inexpensive,portable, and multifunctional systems to perform assays in the field,i.e., outside the laboratory. Applications can include point-of-carediagnostic systems for measuring viral loads, nutritional status,disease biomarkers, or environmental contaminants without the need totransport a sample to a central laboratory. Such tests could beperformed in private residences, global-health facilities, in lawenforcement installations, and medical clinics. The mobile computingdevice can connect to the internet, which will enable combination ofsensor data with patient information and geographical location.Connectivity to an external computation facility can be provided fordata interpretation, geographic and demographic mapping, databaseconstruction and maintenance, and delivery of notifications to remotemedical experts and authorities. Compact, field-operable digitalmeasurement devices will free assays from the requirements for trainedtechnicians in laboratories. Instead, these assays could be performed byanyone, due to the size and affordability of the detection system.

Biosensors

Digital molecular assay systems or devices as disclosed herein compriseelements which may be termed “biosensors.” A biosensor is a device thatuses biological molecules (e.g., one or more enzymes, antibodies, ornucleotide sequences) to detect the presence of chemicals. Many kinds ofbiosensors may be used in a digital molecular assay. A biosensortypically consists of a capture component (often termed the“bioreceptor”) and a reporter component, (“biotransducer”), togethercomprising a reporter molecule as disclosed herein, as well as a systemwhich includes a detector, processor, and display, and optionally otherelements such as a signal amplifier, magnifying lens, and light source.The interaction between (typically the binding of) the analyte and thecapture element produces an alteration in the reporter element, whichoutputs a measurable physicochemical signal. This interaction produces asignal which indicates the presence or concentration of the targetanalyte in the sample.

Reporter components as disclosed herein include optical transducersincluding plasmonic nanoparticles, other localized surface plasmonresonance (LSPR) systems, plasmon scattering systems, photonic crystals,and any other technology that can detect a single molecule and producean optical signal.

Capture elements may be natural or engineered biomolecules, such as anantibody or fragment (Fab, Fv or scFv) or domain (VH, VHH) thereof, or anucleic acid (one or more sections of complementary DNA or RNA to theanalyte of interest).

The capture element is attached to reporter element, e.g. byfunctionalization and layered deposition, or entrapment in a matrix,e.g. a hydrogel or xerogel such as sol-gel. The surface of the sensor towhich reporter molecules (comprising capture and reporter elements) areattached, termed the “reporter surface,” may be, for example, polymer orglass; or glass coated in metal or bearing the metal nanoparticles (e.g.gold or silver; other metals such as titanium, chromium, and copper havealso been used) that comprise the reporter element. This surface formsor is aligned along at least one wall of a chamber or flow cell, into orthrough which the analyte solution is passed.

In an example of a plasmonic nanoparticle biosensor, the chamber or flowcell may be made of glass or polymer; the glass or polymer reportersurface may bear gold nanoparticles functionalized with captureelements, applied via methods known in the art. For analysis with darkfield microscopy, light is passed through the edge of the glass orpolymer reporter surface, orthogonal to the plane of the reportersurface.

Sample Compartment

The measurement device provides a sample compartment suitable forintroduction of a sample of interest. Measurement devices that employoptical measurement techniques will benefit from a sample compartmentthat is oblong, with one short dimension. The light path for the opticalsignal from the reporter molecules to the recording device will alignparallel with the short dimension. This orientation will minimizeabsorption and dispersion of the optical signal that would causeproblems for longer optical paths. This criterion allows for the use ofeither prismatic or cylindrical sample compartments.

Reporter Volume

The sample compartment comprises a component, termed the reportervolume, that contains reporter molecules. This component obviates theneed to add reporter molecules to the sample of interest, and willinstead enable recycling of the reporter molecules. More importantly,the reporter molecules will be held in a substantially stationaryarrangement, so that inactive reporter molecules can be identified andrecorder previous to clinical use of the measurement device.

In some embodiments, the reporter volume is defined by a physicalenclosure that retains reporter molecules within itself. The physicalenclosure may be porous, to allow passage of analyte into the reportervolume for contact with the reporter molecule. In some embodiments, thereporter volume is not defined by a physical enclosure; instead, othermeans can be provided to retain reporter molecules within the reportervolume and keep the reporter molecule stationary.

In certain embodiments, the reporter molecules are associated with athree-dimensional support. In one design, the reporter molecules arecovalently bound to the three-dimensional support. Alternatively, thereporter molecules are not covalently bound to the three-dimensionalsupport, but are impregnated in the three-dimensional support in such amanner as to hinder diffusion from the three-dimensional support. Insuch a design, the reporter molecules are substantially trapped in thethree-dimensional support, and are stationary.

The reporter volume will preferably be narrow in the dimension that isperpendicular to the optical pathway for the reporter molecules. Thisgeometry provides an important advantage: the optical pathway from afirst reporter molecule is unlikely to encounter a second reportermolecule before arriving at the recording device. This is shown in FIG.11 . A narrow reporter volume is shown in FIG. 11(a), with a recordingdevice to the right, and a non-uniform arrangement of reporter moleculesin the reporter volume. It will be seen that, in this geometry, theoptical paths from reporter molecules to recording device are wellseparated. In contrast, a wide reporter volume is shown in FIG. 11(b).In this geometry, at least one reporter molecule overlaps with a secondreporter molecule. This overlap is unfavorable for two reasons: (a) thesecond reporter molecule can partially reabsorb the signal from thefirst reporter molecule, thus causing error, and (b) identification ofinactive reporter molecules, which requires accurate measurement of freeand bound signal from reporter molecules, will be made more complicated.

The degree to which optical paths overlap can be estimated from a smallnumber of parameters that define the receptor volume, including theparticular distribution of reporter molecules (random, semi-random,aggregated, ordered), the concentration and effective size of thereporter molecules, and the thickness of the reporter volume. In certainembodiments of the disclosure, substantially all optical paths betweenreporter molecules and the recording device encounter no other reportermolecule. In certain embodiments, substantially all optical pathsbetween reporter molecules and the recording device encounter at mostone other reporter molecule.

In some embodiments, the reporter volume is sufficiently thin so as toallow for a single layer for the reporter molecules. In this design,overlap of optical paths is not possible, since all reporter moleculesare substantially in a plane perpendicular to the optical path, andparallel to the recording device, as depicted in FIG. 12(a). Variousmonolayer techniques can be used to obtain such a system, such as theuse of surface active molecules. Crosslinking of surface activemolecules can render the reporter molecules stationary. Discussion ofreporter surfaces is presented below in detail.

It should be noted that the preferred distance of closest approach foroptical paths of different reporter molecules is determined not only bythe size of the reporter molecules (which can determine whether anoptical path of one molecule penetrates a second molecule) but also bythe spatial resolution of the detector, and in particular the pixelsize. Ideally, each reporter molecule will be separated by at least apixel from any neighboring reporter molecule, in order to most easilyidentify inactive reporter molecules and observe the optical signal fromactive reporter molecules. Furthermore, depending on the divergence ofthe optical paths that enters the recording device, a much largerseparation may be desirable. This is shown in FIG. 12(b), for which thedistance between reporter volume and recording device has beenincreased, for clarity, and for which a small, but nonzero divergence ofoptical signal that enters the recording device. It will be apparentthat, even though the reporter molecules do not physically overlap eachother, optical signals from closely spaced reporter molecules canpotentially overlap.

In order to minimize overlap between optical paths of different reportermolecules, it will be apparent that features and techniques thatminimize aggregation of reporter molecules or, conversely, increaseseparation between reporter molecules, will be advantageous. In oneembodiment, individual reporter molecules will be coupled to largerparticles, such as microspheres or microparticles, or nanoparticles.Coupling of reporter molecules to larger particles will tend to increasethe average distance between reporter molecules, due to the size of thelarger particles. This is depicted in FIG. 12(c), in which reportermolecules 3 are attached onto, or within, larger particles 5.

Reporter Surface

In certain embodiments, the sample compartment comprises a reportersurface for attachment of reporter molecules. The reporter surface isoriented perpendicular to the shortest dimension of an oblong samplecompartment, in order to minimize the optical path from reportermolecule to recording device. This arrangement of reporter moleculesallows easy attachment of reporter molecules to a solid support, andfurther provides a narrow reporter volume, which is beneficial for thereasons discussed above.

The reporter surface is oriented opposite a window that is substantiallytransparent to the signal produced by the reporter molecules. Thetransparent reporter surface can contact a waveguide that is suitablefor dark-field microscopy. In certain embodiments, the reporter surfacecomprises a metallic layer. In certain further embodiments, the metalliclayer is suitable for surface plasmon resonance. The reporter surfaceand transparent window can be located on the two end faces of aprismatic sample compartment, or alternatively on the two end faces of acylindrical sample compartment. In certain embodiments, the end facesare parallel and proximal, approximating or forming an assay slide orassay chip.

Reporter Molecules

The measurement devices and systems comprise, and the methods disclosedherein employ, a variety of reporter molecules. In one embodiment, asingle type of reporter molecule is employed, which will provide awell-behaved binding response to various concentrations of analytes.Alternatively, two or more different reporter molecules having differingaffinities for the same analyte, can be employed, which can accommodatea larger range of analyte concentration than a measurement device havinga single type of reporter molecule, as described in further detailbelow. In certain embodiments, two or more different reporter moleculeshaving affinities for different analytes are provided.

The reporter molecules may comprise a chromophore. In certainembodiments, the chromophore has been covalently attached to thereporter molecule; alternatively, it may attach to the reporter moleculevia functionalization, e.g. to the surface of a quantum dot or plasmonicnanoparticle. In certain embodiments, the chromophore absorbselectromagnetic radiation. In certain embodiments, the chromophoreabsorbs electromagnetic radiation in a spectral region chosen fromvisible and ultraviolet. Alternatively, the chromophore may scatterelectromagnetic radiation. In certain embodiments, the chromophore isluminescent. In certain embodiments, the chromophore is fluorescent. Incertain embodiments, the chromophore is phosphorescent.

In certain embodiments of the disclosure, the reporter molecules mayeach comprise a chromophore that provides an optical signal upon bindingof analyte. The optical signal may be a change in the extinctioncoefficient of an absorption band, a change in the λ_(max) of anabsorption band, a change in the quantum yield of an emission band, achange in the fluorescence anisotropy of an emission band, a shift inthe center of a spectrum above or below a specified wavelength,wavelength of maximum intensity (λ_(max)), a change in the size orintensity of the signal, an increase or decrease in brightness, a changein the shape of the signal, the presence or absence of spectral bands;and a change in shape of a spectrum.

In certain embodiments of the disclosure, the optical signal is causedby an interaction between analyte and chromophore. In certainembodiments, the optical signal is caused indirectly by the binding ofanalyte to reporter molecule. In certain embodiments, binding of theanalyte by the reporter molecule induces a conformational change thataffects an absorption or emission property of the chromophore. Incertain embodiments, binding of the analyte by the reporter moleculeinduces an interaction between a chromophore on the analyte and achromophore on the reporter molecule.

In certain embodiments of the disclosure, the reporter moleculecomprises two chromophores. In certain embodiments, binding of ananalyte by the reporter molecule induces a geometric change in thereporter molecule that increases a non-radiative interaction between thetwo chromophores. In certain embodiments, binding of an analyte by thereporter molecule induces a geometric change in the reporter moleculethat decreases a non-radiative interaction between the two chromophores.In certain embodiments, the non-radiative interaction is fluorescencequenching. In certain embodiments, the non-radiative interaction isfluorescence energy transfer. In certain embodiments, the non-radiativeinteraction is phosphorescence energy transfer. In certain embodiments,the non-radiative interaction is plasmon-coupled resonance energytransfer.

In certain embodiments, the chromophore is a plasmonic nanoparticleand/or a quantum dot. The plasmonic nanoparticle and/or quantum dot maybe functionalized to bear a capture element. When the capture element isa biological molecule such as an antibody, nucleotide, peptide, orfragment thereof, the chromophore-capture element becomes an opticalreporter molecule, and a biosensor. Contact with (e.g., binding of) ananalyte, such as an antigen or complementary nucleotide, changes themass of the induces a change in nanoparticle's spectral properties, dueto effects such as electron transfer, energy transfer, plasmonresonance, and changes in the particle's mass and mobility.

Inactive Reporter Molecules

The methods described herein accommodate a certain fraction of reportermolecules that is inactive, i.e., the optical signal from these inactivereporter molecules is either absent or substantially different from thebulk of reporter molecules. This behavior can be due to the failure of areporter molecule to bind to the analyte. Alternatively, a reportermolecule can bind to the analyte but does not produce an optical signal,or produces an optical signal that is substantially different from theremainder of optical molecules.

In certain embodiments, the system utilizes a nanoparticle as a reportermolecule. The nanoparticle can become inactive on aggregation with othernanoparticles.

In certain embodiments of the disclosure, an inactive reporter moleculecomprises individual proteins (including antibodies) that haveaggregated, a peptide or protein that has not correctly folded, apeptide or protein that comprises an incorrect residue, a defectivechromophore.

The number of inactive reporter molecules can remain substantiallyconstant during the operating lifetime of the measurement device,particularly in cases in which inactive reporter molecules havedefective composition. It is also possible that the number of inactivemolecules will increase during the operating lifetime of the measurementdevice, due to chemical deterioration of reporter molecules,particularly photochemical deterioration caused by repeated highintensity exposure to light sources, or aggregation of protein formingpart of the reporter molecule.

Inactive reporter molecules can be identified by a change in opticalbehavior: either their failure to produce an optical signal on exposureto analyte molecules, or their production of an optical signal onexposure to analyte molecules that is significantly different from thebulk of the reporter molecules.

In certain embodiments, the plurality of reporter molecules isdistributed randomly. In certain embodiments, the plurality of reportermolecules comprises aggregates of reporter molecules. In certainembodiments, the plurality of reporter molecules comprises a regulargeometric ordering in one or more dimensions. In certain embodiments,each reporter molecule is associated with an exclusion zone, withinwhich no other reporter molecule is located.

Recording Device and Microscope

A recording device is provided to record the optical signal from thereporter molecules. In certain embodiments, the optical signal from thereporter molecules passes through a transparent window of the samplecompartment. In certain embodiments, the recording device will be animage sensor, such as a camera. A CMOS (complementary metal-oxidesemiconductor) camera, for example, is useful because it can read eachpixel individually; additionally, CMOS cameras consume very littlepower, allowing them to last longer when used as part of a device in thefield. Almost all smartphone cameras have CMOS cameras, many withresolution of over 10 megapixels, making them useful in the methods,systems, and devices disclosed herein.

In certain embodiments of the disclosure, the recording device allowsthe observation of one or more signals from the sample compartment. Incertain embodiments, each of a plurality of signals originates from adifferent region of the sample compartment. In certain embodiments, eachsignal in the plurality of signals originates from a pixel in a regulargeometric grid that spans the sample compartment.

In certain embodiments, the pixels of a recording device are arranged ina rectangular or square array. In certain embodiments, the pixels of arecording device are arranged in a 512×512 square array, a 1024×1024square array, a 2048×2048 square array, or a 4096×4096 square array. Incertain embodiments, the signal from each pixel is recorded separatelyfrom all other pixels. In certain embodiments, the signal from 2×2 setsof pixels is binned together.

The measurement device can allow the observation of a plurality ofsignals from different regions of the plurality of reporter molecules.In certain further embodiments, the different regions of the pluralityof reporter molecules are disposed in a regular grid. Alternatively, theindividual optical signal from substantially all reporter molecules canbe observed without interference from any other reporter molecule.

In certain embodiments, the recording device can capture individualpixels and/or individual reporter molecules. The use of plasmonicnanoparticles or quantum dots as substrates to which capture elementsare functionalized facilitates this detection. Used in combination witha magnifying lens, the recording device could detect even smallersignals. Such lenses are well known in the art.

The recording device can use any technique that is known in the art fordetection and quantification of reporter molecule/analyte complexes.Recording devices can use optical absorption and emission methods thatare paired with the reporter molecule design.

The recording device can make optical absorption measurements. Forexample, binding with a reporter molecule can be coupled with anenzyme-linked immunosorbent assay (ELISA) that produces a coloredproduct in the presence of an analyte. When the ELISA is functionalizedonto a plasmonic nanoparticle or quantum dot, the presence and quantityof analyte would then be reported by, for example, the wavelength,intensity, etc. of an absorption feature; and would yield a signal fromeach nanoparticle as opposed to a bulk signal.

Alternatively, the optical output could include fluorescence emissionfrom fluorophore either on the reporter molecule or coupled with thereporter molecule that is excited by a light source. The presence andquantity of analyte would then be reported by the intensity of thefluorescence emission. The fluorophore could be proximal to a surface,such as a photonic crystal, such that the fluorescence emission isenhanced. Multiple fluorophores can be employed to tune the fluorescencesignal to a desirable outcome. Thus, the optical signal can be modulatedby excitation transfer among two or more fluorophores.

Fluorescence and phosphorescence quantum yield, λ_(max) shift, andanisotropy are envisioned in this disclosure. For anisotropymeasurements, polarizers can be introduced into either the excitation oremission optical pathway, or both. A light source can be coupled withthe emission methods. The light source can be a conventional broadbandsource, light emitting diode, or laser, and can be delivered to thesample either directly or via a wavelength selection device such as agrating, in order to optimize excitation. Light can be directed througha total internal reflection component incorporating a waveguide andforming the base of the reporter surface, thus providing dark fieldexcitation.

In some embodiments, the optical assay medium could include a surfaceconfigured for surface-enhanced Raman scattering (SERS). Thus, theoptical output could include Raman scattering of the light source byreporter molecules on the SERS surface. The presence and quantity ofanalyte would then be reported by the intensity of the Raman scattering.

Identification of Inactive Reporter Molecules

Provided herein are methods for identifying inactive reporter molecules,termed “identification method”. For certain systems, two solutions, thefirst free of analyte, and the second with a high concentration ofanalyte, will be contacted sequentially with the reporter surface. Itwill be appreciated that these two solutions will cause an absence ofanalyte binding by reporter molecule, and near saturation of analytebinding by reporter molecule, respectively. Images are recorded usingthe recording device, and a comparison is made between the images forthe analyte-free and analyte-saturated conditions. Reporter moleculesthat do not meet selectivity criteria are marked as inactive.

In the case of inactivation due to nanoparticle aggregation,identification of inactive reporter molecules will be straightforward.Formation of aggregates will be apparent on visual inspection of theimages from the recording device, and will not require the“analyte-free” and “analyte-saturated” procedure outlined above.

The identification method maintains a record for the location ofreporter molecules in the measurement device. The location of reportermolecules can be referenced by x/y coordinates, for example, relative toan appropriate geometric grid in the measurement device, or relative tothe pixel coordinates on the recorder device. The record of inactivereporter molecules can be maintained on non-volatile computer memory.

The identification method will provide criteria for tagging reportermolecules as inactive. The criteria are set to strike a balance betweeneliminating poorly behaving reporter molecules from use, whilemaintaining a sufficiently high count of reporter molecules for theparticular accuracy and sensitivity requirements for the measurementdevice. In order to eliminate bias, and enable automated tagging, anumerical threshold can be chosen, based on the type of optical signalthat is observed. By way of example only, a certain reporter moleculemay undergo a shift in emission λ_(max) on binding to an analytemolecule, and the λ_(max) shifts by 20 nanometers (nm) for the bulk ofthe compounds in this example. A threshold of a 5 nm shift might bechosen for this particular example.

In order to satisfy requirements for accuracy and sensitivity, thenumerical threshold can be chosen in order to exclude a certain fractionof reporter molecules. Referring to the previous example, a λ_(max)shift of 12 nm may be observed for 95% of the reporter molecules. Athreshold λ_(max) of 12 nm may then be chosen in order to retain 95% ofthe reporter molecules as active, and discard 5% of the reportermolecules as inactive.

A variety of criteria can be applied for assigning an inactive status.Importantly, any criteria can be chosen for assigning reporter moleculesas inactive. Since the binding of any one reporter molecule isindependent of all other reporter molecules, elimination of a reportermolecule from the pool of active reporter molecules does not affect thebehavior of the remaining molecules.

If indicated, the identification method described above can be repeatedperiodically during the operating lifetime of a measurement device. Thispractice will be particularly beneficial for reporter devices whoseperformance is susceptible to deterioration over time. Ideally, theidentification method will require a minimal amount of operatorintervention, with the measurement device automatically performing allrequired steps. For the case of nanoparticle-based reporter molecules,images can be recorded periodically, and any aggregation that may occurover time can be identified by pattern-matching software.

The identification method can also comprise the steps of subjecting themeasurement device to one or more solutions containing intermediateconcentrations of analyte. This will be particularly important forquantitative measurement of analyte, for which a range of reportermolecule saturation is envisioned. By use of several solutions, spanninga range of analyte concentrations, a calibration curve can beconstructed to better match optical reporting data with concentration ofanalyte.

A key benefit from the use of spatially resolved signals from a field ofreporter molecules is that regions of the recording device that areparticularly problematic can be flagged as such, and discarded insubsequent analyses. This includes not only cases for inactive reportermolecules, i.e., improperly folded antibodies, but for any region thatpresents difficulties. This may include overlapping spots from two ormore closely spaced reporter molecules, or reporter molecules whose freeand bound states are poorly distinguishable, for whatever reason.Binding of each individual reporter molecule is independent of allothers, and discarding a small set of optical signals can improveaccuracy or precision, while impacting sensitivity only marginally.

Accuracy/Precision/Sensitivity

It is expected that accuracy for the disclosed digital measurementmethods will be at least as good as for conventional analog methods. Thedigital measurement methods will minimize or eliminate several sourcesof error, which by definition is the source of low accuracy. By way ofexample, one source of error arises from reporter molecules which areinactive: either they do not bind to the analyte molecule, or bind tothe analyte molecule and do not provide the expected optical signal.Either situation, if not taken into account, introduces error into theestimation of analyte concentration, since the observed signal will belower than expected.

It is expected that precision for the disclosed digital measurementmethods will be at least as good as for conventional analogmeasurements. Conventional methods, which observe a bulk signal from theentirety of reporter molecules, can provide precision estimates usingvarious statistical and numerical methods in most, but not all cases.

Consider the system such as that depicted in FIG. 3 , which contains acollection of reporter molecules that comprise a chromophore. Theobserved spectral shift of the bulk signal will be the average of allshifts (if any), and may be quite small, for small analyteconcentrations. This shift may be difficult to discern, especiallyconsidering the curve broadening due to the different micro-environmentssurrounding each chromophore.

In contrast, using the digital molecular assay, and observing spectralshifts for each reporter molecule, free reporter molecules will displayzero shift, while bound reporter molecules will display full shift.There is no intermediate state. Naturally, not all chromophores willshift by the same value, but the expected value and range can bedetermined prior to usage in the field. Reporter molecules with outlyingvalues can be discarded as inactive.

It is expected that the signal-to-noise for the disclosed digitalmethods will be at least as good as conventional methods. For bulkdetection, smaller concentrations of analytes will lead to a weaker bulksignal. For digital detection, smaller concentrations of analytes willlead to a smaller number of discrete signals, each of which having thesame intensity or value.

Curve Fitting

The individual signals for discrete reporter molecules, shown assimulations in FIG. 8 , lend themselves to curve-fitting techniques thatcan improve the signal-to-noise ratio. The signal from each activereporter molecule will fall in either of two regions of the spectrum,for which idealized noise-free curves (corresponding to the curves inFIG. 8 ) can be constructed, based on previous knowledge of the reportermolecule. Since each reporter can only be free or bound, the observedsignal from a reporter molecule can be assigned as either free or bound.This stands in contrast to many curve-fitting applications, for whichsuperposition of the signals from two or more states, in varying ratios,must be accommodated in order to model the observed signal.

An example of curve-fitting is shown in FIG. 13 . Emission from twosignals, shown in gray, is summed, with the simulated addition of“noise” to form a net observed signal, shown in black. The signals aresimilar to those presented in FIG. 7 , and the vertical dashed line inFIG. 12 corresponds to the same delineation of analyte-free andanalyte-bound reporter molecule at 500 nm that was discussed in FIG. 8 .Curve-fitting techniques can, given the observed signal, estimate theindividual signals that combined to give the overall signal. Observedsignals that comprise a small number of well-separated individualsignals can be evaluated using curve-fitting with high accuracy andprecision of curve-fitting. Furthermore, because of the digital natureof binding, i.e., the reporter molecule is either bound to the analyteor is free of analyte, a single reporter molecule can provide only oneof two possible signals, corresponding to the analyte-bound andanalyte-free states, with no intermediate state possible. In the exampleshown in FIG. 12 , the observed signal can be clearly attributed to tworeporter molecules, one with emission below 500 nm, and the other withemission above 500 nm, corresponding to an analyte-bound and ananalyte-free reporter molecule. The digital property of this experimentwill substantially simplify the curve fitting process.

In addition, the use of curve-fitting techniques can prove advantageousfor handling two or more reporter molecules whose optical signals cannotbe resolved spatially. In the case of two reporter molecules, whosesignals cannot be resolved from each other, four states are possible:(a) both receptors bound; (b) both receptors free; and (c) & (d): onereporter free (the last two states can be expected to have similar butnot necessarily identical optical signals). This situation isnecessarily more complex than the single reporter molecule; however, itis still very manageable, compared to the optical signal from bulksamples.

Curve-fitting methods can be used for handling two or more reportermolecules whose optical signals are partially resolved spatially, i.e.,the optical signals from the two or more reporter molecules is spreadunequally across a number of pixels. It can be expected that thisscenario will be more common than exact spatial overlap from two or morereporter molecules, especially for recording devices with finely spacedpixels. This scenario can benefit from curve fitting of not a singlespectrum from a single pixel (or a summed spectrum from a collection ofclosely spaced pixels), but instead a collection of individual spectrafrom a collection of closely spaced pixels, combined with profiles forthe (partially overlapping) spots.

Binding Isotherm

The relation between the count of bound reporter molecules and theconcentration of analyte, known as the “binding isotherm”, is complexand indirect. In short, the ratio of bound/total reporter moleculesincreases asymptotically to 1 as the concentration of free analyteincreases. (Generally, the analyte is present in excess, compared to amuch smaller concentration of reporter molecule, so free analyteconcentration and total analyte concentration are approximately equal.This approximation will be used throughout this discussion.) A higheraffinity reporter molecule will bind to a higher proportion of analytemolecule at any given analyte concentration. Importantly, the totalreporter molecule concentration refers only to the active reportermolecule.

Shown in FIG. 14 are two binding isotherm curves. The horizontal axiscorresponds to analyte concentration, and the vertical axis correspondsto the ratio of bound/total reporter molecule. The curved linesrepresent the two binding isotherms, which determine the bound/totalreporter molecule ratio at any given analyte concentration. For eachcurve, the bound/total receptor ratio asymptotically approaches 1 as theanalyte concentration increases, and as the reporter molecule approachessaturation, i.e., most every reporter molecule binds to an analytemolecule. The upper, dark curve corresponds to a higher affinityreporter molecule, and the lower curve corresponds to a lower affinityreporter molecule. It will be apparent that the affinity of reportermolecule for analyte will play a large role in the ease of quantifyinganalytes at different ranges of analyte concentration.

In FIG. 14 , the vertical axis (bound/total ratio of reporter molecule)is the dependent variable, and analyte concentration is the independentvariable, since the bound/total ratio depends on the analyteconcentration. For analytical purposes, the graph is used in reverse;that is, the bound/total ratio is obtained from the experiment, and islocated on the vertical axis. From the graph and the binding isothermcurve, the corresponding free analyte concentration is found on thehorizontal axis. Visually, this process can be understood by drawing ahorizontal line from the observed bound/total ratio on the y-axis to theisotherm curve, and dropping a vertical line to the x-axis, in order tofind the analyte concentration. (In practice this process is donemathematically or numerically; however the error analysis still holds.)

FIG. 15 corresponds to a sample with a free analyte concentration ofapproximately 0.10, which corresponds to a bound/total ratio ofapproximately 0.72. This condition is indicated on the binding isothermas a solid circle. A “correct” determination of 0.72 for bound/totalratio, on the vertical axis, is traced horizontally to the bindingisotherm, and down to the horizontal axis. This process is indicated bytwo black arrows. The result of underestimating the bound/total ratio byabout 5% is shown with two gray arrows, with a corresponding grey circleon the binding isotherm.

FIG. 16 corresponds to a sample with a higher free analyte concentrationof approximately 0.40, corresponding to a bound/total ratio ofapproximately 0.92, and again marked with a solid circle on the bindingisotherm. A correct determination is indicated with two black arrows, aswith the previous example. Underestimation of the bound/total ratio by5% is again shown with gray arrows and gray circle. In this example, a5% underestimation in bound/total ratio leads to a substantial error ofabout 40% in analyte concentration. The difference in behavior betweenFIG. 15 and FIG. 16 is due to the different slope of the bindingisotherm graph at the two spots depicted in these two graphs, and thiserror propagation will be more serious at higher concentrations ofanalyte, where the binding isotherm curve is flatter.

Analytical devices that utilize molecular binding for quantification ofanalytes are susceptible to another source of error: in many cases, notall reporter molecules are active for binding of analyte. This causeserror in estimation of the bound/total ratio which, as outlined above,can propagate into substantial error in the estimated analyteconcentration.

By way of example, consider a measurement device in which 10% of thereporter molecules are inactive. Saturation of reporter molecules willproduce only 90% of the expected maximal signal, since 10% of thereporter molecules fail to provide an optical signal (either due tofailure to bind analyte, or failure of the receptor/analyte complex toproduce an optical signal). Inspection of FIGS. 15 and 16 will revealthe substantial error that will be introduced into measurements,particularly at higher analyte concentrations. To some extent, thiserror can be addressed by pre-screening measurement devices undersaturation conditions to estimate the maximum signal, to be used as abenchmark for future measurements. However, this procedure is not ideal,since full saturation can never be accomplished.

Shown in FIG. 14 are two binding isotherm curves. The upper, dark curvecorresponds to a higher affinity reporter molecule. The advantage ofusing this reporter molecule is that nearly complete saturation can beachieved at relatively low concentration: at 0.40 nM, the reportermolecule is 90% saturated. The disadvantage is that this reportermolecule is useful for quantifying smaller concentrations of about 0.10nM, since the shallow region of the binding isotherm curve is subject tosignificant error, as discussed above.

In contrast, the lower, light curve, which corresponds to a loweraffinity reporter molecule, is useful to quantify a larger range ofsample concentrations, since the curve is relatively steep for theentire range. However, the reporter molecule achieves at most 70%saturation in this range, and determination of the signal correspondingto 100% saturation will be difficult to achieve.

The digital molecular assay methods disclosed herein can include thestep of sample dilution, which will improve the precision of estimatedanalyte concentration by reducing the susceptibility of the measurementto errors in bound/total reporter molecule concentration. By way ofexample, consider the sample discussed above, whose binding behavior isdepicted in FIG. 15 . Because of the high degree of reporter moleculesaturation, the binding isotherm curve is very shallow in the regionaround the analyte concentration of 0.40, and determination of analyteconcentration is therefore very susceptible to even small errors inbound/total reporter molecule concentration. This ratio is made moreprecise by digital measurements, as discussed above; however, it wouldbe preferable, from the outset, that the determination of analyteconcentration not be as susceptible to errors in bound/total reportermolecule concentration.

Consider the effect of a four-fold dilution of this sample, i.e.,addition of sufficient volume of solute in order for the concentrationof analyte to drop from 0.40 to 0.10. As a result of this dilution, thebinding behavior would now be represented by FIG. 14 . Because of thesteeper binding isotherm curve in the region around the new analyteconcentration of 0.10, determination of analyte concentration is muchless susceptible to errors in bound/total reporter concentration.

The sensitivity properties of digital measurements make the step ofsample dilution an appealing procedure for improving precision. It willbe readily apparent that the precision of a bulk “analog” measurement ofbound reporter molecule concentration will suffer upon sample dilution.Going from FIG. 14 to FIG. 13 , bound reporter molecule concentrationdrops from 0.92 to 0.72, representing a 22% drop in bound concentration.A bulk analog signal that is proportional to bound reporter moleculeconcentration would drop in magnitude by 22%. This drop in magnitudewould almost certainly be accompanied by an increase in signal-to-noise,since the magnitude of noise would remain constant. Therefore, theincrease in precision due to the binding isotherm effect described abovewill be counteracted by a decrease in precision due to worsenedsignal-to-noise in the determination of bound reporter concentration.

In contrast, digital measurements are much less sensitive to thisdeterioration of signal-to-noise with dilution. As discussed above, adecreased count of bound reporter molecules affects digital measurementsdifferently than analogue measurements. Rather than weakening the bulksignal of analog measurements and thus worsening signal-to-noise, adecreased count of bound reporter molecules will simply reduce the countof discrete optical signals that are received by the reporter device.Importantly, the intensity of each of these discrete optical signalswill remain unchanged.

For this reason, dilution methods to improve precision as describedabove are not accompanied by a decrease in precision due to analogsignal-to-noise effects, and will therefore prove beneficial for digitalmeasurements. The measurement methods can include a step of pre-dilutinga sample before introduction into the measurement device. Alternatively,the measurement methods can, after reporting an analyte concentration,indicate to the user that a repeat measurement of the sample wouldimprove precision, and can further advise the user on a recommendeddilution level.

The measurement methods can include a rapid pre-screen of a sample thatis optimized to quickly provide a low-precision estimate of analyteconcentration, in order to suggest an optimal dilution.

In many uses that are envisioned for the disclosed methods, thresholdsor cutoffs have been established that correspond to critical values.These thresholds or cutoffs can correspond to regulatory levels set byenvironmental laws, or to critical biomarker levels that correspond tocertain health conditions. The measurement methods can adjust therecommended scan parameters and dilution level in order to providemeasurement conditions that are adequate for the intended use.

In certain embodiments, the measurement device can provide a mechanismfor the automatic dilution of a sample. This can be accomplished byejecting a fraction of an existing sample, followed by introduction ofsolute for dilution. This can also be accomplished by introducing a newsample that has been pre-diluted with solute.

In certain embodiments, the recommended dilution level can be calculatedby a computing device, either incorporated into the measurement deviceor connected to the measurement device. The computing device can providethe recommended dilution level to the operator via an interface (such asa display, printout, or synthesized voice report). The computing devicecan also directly control the measurement device to perform any stepsneeded to automatically analyze a diluted sample, without the need foruser intervention.

In certain embodiments, the thresholds or cutoffs can be pre-set in thecomputing device, either in the form of firmware, which can beoptionally updated in the case a threshold or cutoff changes, or in theform of software. In addition, the operating software can prompt theuser for further input on the sample that is being measured. Forexample, in the case of the measurement of biomarkers, the user caninput history parameters for a subject, such as age, weight, gender, andthe like, that may alter a threshold or cutoff and that may thereforeinfluence the precision that is required for a given measurement.

EXAMPLES

The invention is further illustrated by the following examples.

Example 1: Plasmonic Sandwich Immunoassay

In one implementation of the digital molecular assay in a dual antibodyimmunoassay format is described here. Dual antibody immunoassays arewidely used in classic clinical assays, and standard antibody pairs forthis application can be readily obtained. For detection, plasmoncoupling between metal nanoparticles, which will become linked via theanalyte in the sandwich immunoassay, provides a robust readout ofbinding of the analyte molecule to the particles. Plasmon couplingoffers a great strength in that the scattering wavelength of the coupledparticles can differ substantially from the individual particlescattering wavelengths, leading to distinct color changes easilydiscerned at the single particle level by color—even on a cell phonecamera.

In this example, spherical gold nanoparticles (e.g. between 10 nm and100 nm in diameter) can work well. For the present purposes, label freedetection is unnecessary and detection is achieved by the secondarymetal nanoparticle as a signal enhancer. This involves the binding ofanother nanoparticle (gold) through a second antibody resulting in aplasmonic coupling between the two nanoparticles leading to markedspectral shifts. This strategy has been utilized in the detection ofconformational changes in proteins and DNA molecules and will enableeasy detection of single analyte binding in images acquired using simplecameras, such as the ones available on cell phones. The enhanced colorchange due to the second nanoparticle depends on the size of the secondnanoparticle as well as the effective distance between thenanoparticles. The interparticle separation (antibody I-analyte-antibodyII) expected in our assay will be 20-30 nm and is within the limits ofthe effective plasmonic coupling. While we typically use 40 nm goldnanoparticles, the dependency of the exponential decay in the plasmoniccoupling with interparticle distance on the size of the secondnanoparticle (i.e. smaller nanoparticles show rapid decrease inplasmonic coupling compared to larger ones) further allows fine tuningof the spectral shift by using large gold nanoparticles.

Dark-field imaging is one appropriate way to monitor the scatteringlight from individual particle pairs in the assay. Dark-field microscopyis an optical technique wherein the sample is not directly illuminated.Instead, scattered light is used for the visualization of objects thatresults in a near-black background intensity leading to a greatlyenhanced contrast between objects and background. Indeed, nanoplasmonicmaterials yield large number of photons without blinking orphotobleaching, phenomena that mar fluorescence-based detection that iscommonly used in conventional biosensing assays, thus enablingobservation of individual particles with a simple optical setup.

The key to the digital molecular assay is individual evaluation of eachparticle in the field of view. This can be achieved in various ways, butone way involves before and after image comparison, in which the captureparticle is first arrayed (randomly or in patterns) on a substrate. Thearrayed particles are imaged before introduction of sample providing the‘before’ information. The sample is next flowed into the chamber alongwith the secondary labeled antibody. In a successful analyte capture, asecondary label particle will be associated with the capture particleleading to a defined change in brightness and color. Each particle inthe before image will be characterized to determine that it has theexpected brightness and color of single capture particles. Anyaggregated or otherwise altered particles will be ignored in subsequentanalysis. After analyte capture, all images of objects at the locationsof good capture particles are analyzed. A statistical criterion forimage interpretation is utilized to distinguish failed (e.g. aggregatedor nonspecifically bound particles) from clean analyte captures.Background analysis and error correction is enhanced by includingnon-functionalized particles as fiducial markers. These can be used tosense successful infiltration of solvent, air exposure, or any of avariety of other failure modes that can renders portions of the particlefield unusable. In bulk assays, these sorts of errors simply degrade thesignal. But in the digital molecular assay format, such errors can beremoved ahead of time. In fact, in many single molecule fluorescenceimaging experiments it is common for large regions of the field of viewto be unusable for various reasons, but with plenty of good molecules inbetween, the experiments can still run successfully.

Example 2: Plasmonic Hybridization Assay

In a modification of the above example, hybridization of surface-boundnucleic acid probes is combined with plasmonic coupling betweennanoparticles to provide a digital molecular assay for nucleic acidanalytes. A surface is modified with a first oligonucleotide to whichhas been attached a first nanoparticle. The surface modification can beperformed by noncovalent adsorption, or by covalent binding. Thesequence of the first oligonucleotide is chosen to hybridize with afirst complementary sequence that is contained in the desired analytenucleic acid. Exposure of the modified surface then induceshybridization of the analyte with the first short oligonucleotidesequence. At this point, a second oligonucleotide, to which is added asecond nanoparticle, is introduced. The sequence of the secondoligonucleotide is chosen to hybridize with a second complementarysequence that is contained in the desired analyte nucleic acid, with thefirst and second complementary sequences sufficiently separated fromeach other to allow simultaneous hybridization of both the first and thesecond oligonucleotide sequences.

The supramolecular assembly caused by the hybridization of the analytenucleic acid with the first and the second oligonucleotide sequences canbe designed to bring the first and second nanoparticles into closeproximity, as for the sandwich immunoassay described above. Although notstrictly necessary for the successful design of this system, adoption ofcanonical helical structures by the hybridized analyte nucleotide willsimplify the molecular geometry, and facilitate the choice of suchparameters as sequence length and the nature and placement of attachmentto nanoparticles.

All references, patents or applications, U.S. or foreign, cited in theapplication are hereby incorporated by reference as if written herein intheir entireties. Where any inconsistencies arise, material literallydisclosed herein controls.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1-43. (canceled)
 44. A digital assay system for determining aconcentration of analyte in a sample, comprising: a microprocessor;memory; image analysis software stored in the memory and comprisinginstructions being executable by the microprocessor to perform a method,the method comprising: analyzing an image of a plurality of signalsemitted by at least one type of optical reporter molecules incubatedwith at least one type of analyte molecules, and digitally classifyingthe plurality of signals as active or null; and optionally, acommunication interface. 45-65. (canceled)
 66. The digital assay systemof claim 44, wherein the image of a plurality of signals is sent to avirtual server in a computer cloud.
 67. The digital assay system ofclaim 44, wherein each of the optical reporter molecules is spatiallyresolvable.
 68. The digital assay system of claim 44, wherein theoptical reporter molecules comprise plasmonic nanoparticlesfunctionalized with a capture element.
 69. The digital assay system ofclaim 68, wherein the capture element is chosen from: one or morenucleotide sequences that binds the analyte; and an antibody or afragment thereof that binds the analyte.
 70. The digital assay system ofclaim 69, wherein the analyte is chosen from an antigen and a nucleotidesequence.
 71. The digital assay system of claim 70, wherein the captureelement is chosen from an antibody, or a fragment thereof, and one ormore nucleotide sequences complimentary to the analyte nucleotidesequence.
 72. The digital assay system of claim 66, comprising acommunication interface.
 73. The digital assay system of claim 72,wherein the communication interface is wireless.
 74. The digital assaysystem of claim 73, further comprising an image sensor.
 75. The digitalassay system of claim 74, wherein the image sensor is configured tooperating as part of a dark-field microscope.
 76. The digital assaysystem of claim 74, wherein the image sensor is chosen from a megapixelcamera and a complementary metal-oxide semiconductor camera.
 77. Thedigital assay system of claim 74, further comprising a source of lightor other electromagnetic radiation.
 78. The digital assay system ofclaim 77, wherein the source of light or other electromagnetic radiationis a light-emitting diode.
 79. The digital assay system of claim 78,further comprising a sample chamber that is optionally removable. 80.The digital assay system of claim 79, additionally comprising: areporter surface made of glass or polymer, to one side of which opticalreporter molecules comprising plasmonic nanoparticles functionalizedwith capture elements have been affixed, optionally in a grid or anapproximation thereof; and a waveguide that is suitable for dark-fieldmicroscopy in contact with the opposite side of the reporter surface.81. The digital assay system of claim 80, wherein each affixed opticalreporter molecule is resolvable as one pixel of a recording device. 82.The digital assay system of claim 81, wherein the microprocessor,memory, image sensor, software, screen capable of displaying an image,and communication interface are all comprised within a single, portabledevice.
 83. The digital assay system of claim 82, wherein the single,portable device is chosen from a smartphone and a tablet computer. 84.The digital assay system of claim 83, additionally comprising a case forpositioning the portable device, sample chamber, and light source inclose and stable proximity to each other.